Patterned cardiomyocyte culture on microelectrode array

ABSTRACT

The invention provides a culture of patterned cardiomyocytes, the culture including a support substrate bearing a multielectrode array, a negative surface resistant to cell attachment and deposited on the support substrate covering the multi-electrode array, a pattern ablated on the negative surface, a positive surface promoting cell attachment deposited on the pattern ablated on the negative surface; and cardiomyocytes adherent to the positive surface and growing aligned along the pattern ablated on the negative surface. The invention also includes a method of making the culture of patterned cardiomyocytes and a method of in vitro method of testing the effect of a compound on cardiac function by measuring electrical output from the patterned cardiomyocytes.

RELATED APPLICATION

This application claims priority from provisional application Ser. No.61/257,504, filed on 3 Nov. 2009, which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under agencycontract/grant number R01 EB005459 and K01 EB003465 awarded by theNational Institutes of Health. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates to the field of new drugs and, moreparticularly, to a patterned culture of cardiomyocytes formed on amultielectrode array and useful for in vitro testing for possiblecardiac side effects by a new drug.

BACKGROUND OF THE INVENTION

The development of a high-throughput, high-information content device tostudy and understand cardiac electrophysiology would be important forthe fields of cardiac physiology, tissue engineering and drug research.More than 850,000 people are hospitalized for arrhythmias each year andventricular fibrillation (VF) is a leading cause of cardiac death[1].Despite the intensive research in this area, the mechanism of VF isstill poorly understood[2-5].

Arrhythmia is a known side effect of commercial drugs. One of themechanisms by which drugs can cause a potentially fatal form ofventricular tachy arrhythmia, called Torsades de pointes (Tdp), isthrough the prolongation of the QT interval (in an ECG the length of theventricular action potential). It has been reported that approximately2-3% of all prescribed drugs can cause long QT syndrome[6, 7]. A broadrange of cardiovascular drugs and antibiotics also have the potentialrisk of causing drug induced Tdp[8, 9]. At the same time, prolongationof the QT interval does not necessarily lead to Tdp; lengthening of theQT interval could even be antiarrhythmogenic, as it is considered amechanism of action of the class III anti-arrhythmics[8, 9]. Thus, arelatively high-throughput method to identify cardiac side effects anddifferentiate between arrhythmic and anti-arrhythmic effects at an earlystage of drug development would have a significant impact on the field.

Gap junctions play an important role in the propagation of excitation incardiac tissue. Changes in gap junction function affect major cardiacparameters, such as conduction velocity (CV). It has been observed inseveral cardiovascular diseases that the expression of connexins(protein molecules that form gap junction channels) is decreased ortheir distribution is changed, leading to a malfunction in gap junctioncoupling[10]. Understanding the pharmacological modulation of cardiacgap junction channels would further aid the drug development enterprise.

Introduction of an in vitro method for cardiac side effect testing,which has high predictive value, would have a significant impact on drugdevelopment as it could also reduce the cost, time and the number ofdrugs failing in clinical trials[11]. in vitro testing would also reducethe need for animal testing and could be used to study drug effects witha functional assay, but at the cellular level. Other in vitro methods,such as whole heart experiments (Langendorff heart model) or thePurkinje fiber preparation are difficult and time consuming[11].Traditional methods used to study QT interval prolongation at thecellular level include patch-clamp experiments. However, theseexperiments are time intensive, require a skilled operator and cannot beused to study action potential (AP) propagation or parameters such as CVand re-entry. Moreover, evidence suggests that prolongation of QTintervals is not the best predictor of Torsades de pointes. Themeasurement of the length, or the variability in the length, of therefractory period after a cardiac action potential may have morerelevance for predicting arrhythmic behavior[9].

Cardiac myocytes cultured on microelectrode arrays (MEA) have severalbenefits compared to either traditional patch clamp electrophysiology orisolated organ methods. The use of MEAs in the investigation of cardiacside effects would provide information in a relatively high-throughputand low cost manner compared to standard patch-clamp electrophysiology.However, at this time, it is still a low information content method andthis has limited its use. Cardiac myocytes on MEAs have been used in anumber of studies to investigate the effect of toxins, such aspesticides[12] and cardioactive drugs[13] on cardiac field potentials. Acommercial system has also been introduced to measure QT intervals in arelatively high-throughput fashion[14], but, to date, it has onlylimited applications. However, cardiac myocytes can now be maintainedover longer periods of time[15], thus chronic experiments, such as themonitoring of network remodeling for specific diseases, is now feasible.In addition, serum-free formulations for cardiac culture have also beenintroduced, which would increase the reproducibility of such asystem[16].

All of the above mentioned studies utilized unorganized monolayers ofcardiomyocytes on the MEAs. Development of a patterned cardiac myocytelayer that is aligned with the electrodes of a MEA could solve severalproblems associated with the random spread of excitation in a cardiacmonolayer, which makes evaluation of the obtained data, such as CV,difficult. It would also enable the development of specific open-loop orclosed-loop stimulation protocols to measure critical parameters, suchas the length of the refractory period after the action potential. Itcould also be used to create a high-throughput, low-cost functionalreentry model.

There are several lines of evidence indicating that not only contactinteraction with the surface but the shape of the attachment areadetermines the physiology of cardiac myocytes[17]. Pattern geometriesdetermine the extent of the alignment/of the long axis of cardiacmyocytes, alignment determines CV[18] and other physiological andpharmacological properties of cardiac tissues[19, 20].

Several different methods have been developed for cell patterning. Onecategory of this technique is based on direct placement of cells orextracellular matrix molecules on desired locations and includespatterning through microfluidic channels[21-23], microcontactprinting[24, 25] and inkjet printing[26]. Cardiac myocytes havepreviously been patterned on glass using photoresist[27] as well asother techniques[15, 17, 19, 20, 25]. Another method utilizedphotolithography following surface modification with self-assembledmonolayers (SAMs) for neurons[28-30] as well as myocytes[15, 31]. Thebenefit of this method is the compatibility of the technique with cheapautomated silicon manufacturing steps and the ability of the cells toself-assemble after random plating.

SAMs are one molecule thick monolayers attached to a surface composed oforganic molecules, which have been extensively used for surfacepatterning[31-33]. Surface modification with SAMs is also compatiblewith advanced photolithography methods[30, 34]. Studies have also shownthat cells survive on these surfaces for extended periods of time[35,36], do not migrate off the patterned areas[34] and exhibit the typicalmorphology and physiology of the specific cell type[16, 37].

The goal of this study was the development of patterned, rat,cardiomyocyte cultures on MEAs in a serum-free medium for the study ofcardiac physiology and pharmacology utilizing a high-throughputtechnique, but with high information content. An adsorbed fibronectinlayer was used as the foreground because it supported cardiac myocyteattachment and growth and a2-[Methoxy(Polyethyleneoxy)Propyl]TrimethoxySilane (SiPEG) SAM was usedas the cell repellent background because of its excellent proteinadsorption resistant properties[38]. The measurement of CV with thepatterned cardiac myocyte monolayers and the feasibility to applydifferent stimulation protocols to the MEA/cardiac system wasdemonstrated. The action of 1-Heptanol and Sparfloxacin was alsoassessed. This method could easily be adapted for use with human cardiacmyocytes to eliminate interspecies differences in drug side effectscreening and could become an alternative to the existing more complex,expensive and time consuming methods.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a culture of patterned cardiomyocytes. The culture comprises asupport substrate bearing a multielectrode array and a negative surfaceresistant to cell attachment and deposited on the support substratecovering the multi-electrode array. The negative surface bears a patternablated thereon, preferably by laser photolithography. A positivesurface promoting cell attachment is deposited on the pattern ablated onthe negative surface and cardiomyocytes adherent to the positive surfaceand growing are aligned along the pattern.

Preferably, in the culture of patterned myocardiocytes the negativesurface comprises a material selected from the group consisting ofpolyethylene glycol, polyacrylic acid and polyacrylamide. Mostpreferably, the material selected is polyethylene glycol and it isdeposited essentially as a monolayer. The pattern on the negativesurface is preferably ablated by laser lithography through a mask, asfurther explained below.

The positive surface preferably comprises a material selected from thegroup consisting of fibronectin, trimethoxysilylpropyldiethylenetriamineand pure glass.

In the culture described the cardiomyocytes are preferably of mammalianorigin, and may be of neonatal rat Additionally, however, thecardiomyocytes may be derived from human embryonic stem cells.

The invention further includes a method of making the described cultureof patterned myocardiocytes. The method comprises preparing a supportsubstrate bearing a multi-electrode array, overlaying on the supportsubstrate a negative surface resistant to cell adherence thereon,comprising polyethylene glycol and covering the multi-electrode array,ablating a pattern on the negative surface, depositing on the ablatedpattern a positive surface promoting cell adherence thereon andincluding fibronectin, adhering cardiomyocytes on the positive surface,and culturing the cardiomyocytes so that they grow on the positivepatterned surface and aligned therewith.

Yet additionally, the invention also includes an in vitro method oftesting the effect of a compound on cardiac function. This methodcomprises providing patterned spontaneously beating cardiomyocytesaccording to the described culture. Recording a first electrical outputfrom the cardiomyocytes. Contacting the cardiomyocytes with the compoundbeing tested, Recording a second electrical output from thecardiomyocytes after being contacting with the compound. Finally,determining a change between the first and second electrical outputs asan indicator of altered cardiac function.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings. Thedrawings are presented solely for exemplary purposes and not with intentto limit the invention thereto. The following descriptions apply.

FIG. 1: Patterned neonatal cardiomyocytes. A: Pattern design anddimensions. B,C: Phase contrast pictures on day 12 of different regionsof the pattern indicated in A. Field potentials measured at individualelectrodes are marked in red in B. Electrode distance: 200 μm, D:Immunostaining with Rhodamine phalloidin for F-actin indicating cardiacmyofibrils (note the visible striations). Nuclei are stained with DAPI(blue).

FIG. 2: Action potential (AP) conduction in patterned cardiac myocytemonolayers. A: phase contrast picture of cardiac myocyte patterns on thetop of the substrate embedded electrodes (electrode distance is 200 μm)with a sketch of the conduction pathway on the completed pattern andwith the recorded extracellular signals on the given electrodes. B:Overlay of the recorded traces showing the temporal relationship of thesignals. A spontaneous AP was generated in the cardiac myocyte monolayerin the right fork of the pattern, close to electrode 74. This AP spreadthrough the pattern reaching electrode 74 first, electrode 72 second andwith a much longer delay (due to the longer path) electrode 52 last.Conduction velocity (CV) was determined based on the time delay in thesignal between electrode 74 and 52 and the known pattern geometry. Thecalculated CV in this experiment was 0.190.

FIG. 3: Measurement of conduction velocity in patterned cardiac myocytemonolayers with electrical stimulation. A: Electrodes 52 and 84 werestimulated alternatively with a time delay of 300 ms and the responsewas recorded at electrode 72. B: E72 exhibited both long and short timedelays in FP generation based on the distance the AP had to travel fromthe stimulation site.

FIG. 4: Measurement of refractory period after cardiac action potential.Electrodes 63 and 85 (see pattern geometries on the left) werestimulated alternatively according to the following protocol: E63, 100ms delay, E85, 400 ms delay, E63 again. Right panel shows recording atelectrode 87. Stimulating E85 100 ms after the stimulation of E63 failedto evoke an AP because this delay was too short, cells were stimulatedin their refractory period. By chance, cell electrode coupling onelectrode E85 was especially strong in this experiment, thus theextracellular recording approximately replicated the shape of theintracellular AP illustrating the relationship of the AP and thestimulation. When the stimulation delay between E63 and E85 was 250 ms,both stimulations evoked an action potential at electrode 87.

FIG. 5: Effect of the gap junction blocker 1-Heptanol. A: Propagation ofexcitation between the two end points in the pattern (Electrodes 84 and52) before addition of the drug. The CV was measured to be 0.197 m/secB: Propagation of excitation after the drug. The CV was calculated as0.0066 m/sec. The drug effect on the CV measured over 4 MEAs.

FIG. 6: Effect of the HERG channel antagonist Sparfloxacin. A: Fieldpotential recordings before addition of Sparfloxacin indicated stablebeating frequencies and synchronous activity B: Field potentialrecording after the addition of 2 μM Sparfloxacin showed burst likeactivity with higher intra-burst frequencies and no synchrony betweenelectrodes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown.

Unless otherwise defined, all technical and scientific terms used hereinare intended to have the same meaning as commonly understood in the artto which this invention pertains and at the time of its filing. Althoughvarious methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. However, the skilledshould understand that the methods and materials used and described areexamples and may not the only ones suitable for use in the invention.

Moreover, it should also be understood that any temperature, weight,volume, time interval, pH, salinity, molarity or molality, range,concentration and any other measurements, quantities or numericalexpressions given herein are intended to be approximate and not exact orcritical figures unless expressly stated to the contrary. Accordingly,where appropriate to the invention and as understood by those of skillin the art, it is proper to describe the various aspects of theinvention using approximate or relative terms and terms of degreecommonly employed in the art, for example, so dimensioned, about,approximately, substantially, essentially, consisting essentially of,comprising, and effective amount.

Further, any publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety as if they were part of this specification. However, in case ofconflict, the present specification, including any definitions, willcontrol. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting.

Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough, complete, and will fully convey thescope of the invention to those skilled in the art. Other features, andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

Materials and Methods

Surface Modification of Microelectrode Arrays with PEG Silanes

MEAs containing 60, 10 μm diameter electrodes (Multichannel Systems,Germany) were cleaned by soaking the arrays in a detergent solution for2 hours followed by sonication for 10 minutes. The arrays were thenoxygen plasma cleaned for 20 minutes. Surface modification was completedby incubation of the MEAs in a 3 mM PEG silane,2-[Methoxypoly(ethyleneoxy)propyl]trimethoxysilane (MW=460-590, Gelest),solution in toluene, with 37% concentrated HCL added to achieve a finalvalue of 0.08% (0.8 ml HCL/L), for 45 minutes at room temperature. Thearrays were then rinsed once in toluene, twice in ethanol, twice inwater and sonicated in water for 2 minutes to remove the non-covalentlylinked material[39]. The arrays were air dried with nitrogen and storedin a dessicator overnight.

Laser Ablation and Patterning of the Microelectrode Arrays

The MEAs were patterned by laser ablation photolithography using a deepUV (193 nm) excimer laser (Lambda Physik) at a pulse power of 230 mW anda frequency of 10 Hz for 45 seconds through a quartz photomask(Bandwidth foundry, Eveleigh, Australia). The arrays were sterilizedusing 70% isopropanol and then incubated with 5 μg/ml of fibronectin ina Phosphate buffered solution (Invitrogen) for 20 minutes at roomtemperature. The solution was removed and the surface was first rinsedwith PBS, followed by the plating medium, and then dried before thecells were plated.

Neonatal Rat Cardiomyocyte Culture

The neonatal rat cardiomyocyte culture was prepared using the cardiacisolation kit from Worthington[40]. All animal work was approved by theUCF IACUC and followed NIH guidelines. Briefly, two day-old rat pupswere euthanized in a precharged CO₂ chamber. Hearts were dissected andminced in ice cold Hanks balanced salt solution (HBSS). Cardiac myocyteswere dissociated by incubation of the hearts in trypsin (100 μg/ml inHBSS) for 16 hours at 2-8° C. The hearts in the trypsin solution werebriefly warmed with a trypsin inhibitor before adding collegenase (300units/ml in L-15 medium) for 45 minutes in a water bath at 37° C.followed by mechanical trituration. The cell solution was filtered toremove any remaining tissue and centrifuged at 50 g for 5 minutes at 22°C. The cells were resuspended in high glucose Dulbecco's modified eaglemedium (DMEM, Gibco/Invitrogen) supplemented with 10 ml Fetal BovineSerum (Gibco/Invitrogen) and 1 ml penicillin streptomycin(Gibco/Invitrogen), preplated in Petri dishes and incubated at 37° C.and in 5% CO₂ for 45 minutes. This was necessary to eliminate thefibroblasts. The supernatant from the Petri dishes was centrifuged at 50g for 5 min at 22° C. The cells were then resuspended in the platingmedium. The serum-free plating medium consisted of: 100 ml Ultraculturemedium (Bio Whittaker Cambrex) supplemented with 10 ml B27, 1 mlL-glutamine (Gibco/Invitrogen), 1 ml Penicillin Streptomycin, 0.375 gdextrose (Fisher Scientific) in 800 μl water, 1 ml non-essential aminoacids and 1 ml of Hepes buffer (Gibco/Invitrogen)[41]. Additional growthfactors were also added to improve cell survival in the serum-freeconditions. They included 0.1 μg/ml of L-thyroxine, 10 ng/ml ofEpidermal growth factor (Sigma-aldrich) and 0.5 μg/ml of Hydrocortisone(BD biosciences). Cells were plated at a density of 1000 cells/mm² onthe MEAs. The medium was changed 24 hours after plating. Subsequentchanging of the medium was performed every third day.

Immunostaining

Patterned Cardiomyocytes were immunostained for F-Actin with RhodaminePhalloidin (Invitrogen, R415), using a protocol provided by the company.Briefly, the cells were washed with PBS and fixed using 3% Formaldehyde.The coverslips were extracted with 0.1 ml Triton X®. The stainingsolution (with 1% Bovine Serum Albumin to prevent background staining)was added at a dilution of 1:40 in PBS and coverslips were incubated for30 minutes. Imaging was done using confocal microscopy.

Multielectrode Extracellular Recordings

The cardiac myocytes were cultured on patterned metal MEAs (Planar 10 μmelectrodes, 200 μm separation, Multichannel-systems). A 60 channelamplifier (MEA1040, Multichannel-systems) was used to record electricalactivity from the spontaneously beating cardiac cells. The sameelectrodes were also used for stimulation utilizing a stimulus generator(STG 1002, Multichannel systems). The cells were stimulated utilizing500 mV, 1 ms wide bipolar pulses at 2 Hz. The recording medium was thesame as the plating medium with the pH adjusted to 7.3 using HEPESbuffer. After a 30 minute incubation period, APs were detected andrecorded using built in functions of the Multichannel System software.For drug experiments, 50 μM 1-Heptanol (Gibco/Invitrogen) was added tothe bathing medium and recordings were performed before and 15 minutesafter drug administration with additional recordings done at 15 minuteintervals. For Sparfloxacin (Sigma-Aldrich), 2 μM of the drug was addedto the recording medium and recordings were taken in 15 minute intervalsbefore and after drug administration. The data was further analyzedusing software written using Matlab and Clampfit (Axon Instruments).

Results

Surface Modification of the Microelectrode Arrays

Our laboratory routinely uses SAMs to modify and pattern glasscoverslips for cell culture applications[15, 16, 30-32, 42]. However,patterning cardiac myocytes on MEAs presented a challenge for threeprimary reasons: 1) The complex composition of the MEA surface made theverification of the surface modification step difficult, 2) MEAs areexpensive, thus methods needed to be developed that enabled them to becleaned and refunctionalized for repeated use and 3) patterned cardiacmyocytes do not grow optimally on the standardtrimethoxysilylpropyldiethylenetriamine (DETA) surfaces[15] nor on cleanglass. Thus, a cell resistant background surface was needed which wasalso resistant to protein adsorption as this would allow fibronectinincubation on the foreground and prevent its adherence to thebackground, enabling cardiac myocyte attachment. Thus, poly(ethyleneglycol). (PEG) was chosen for the background because of its excellentprotein adsorption resistance. X-ray Photoelectron Spectroscopy (XPS)and contact angle measurements were used to analyze the results of thesurface modifications and throughout the entire study for qualityassurance purposes.

Cultured Neonatal Rat Cardiomyocytes on Patterned Surfaces in Serum-FreeMedium

The growth and activity of neonatal rat cardiomyocytes on the patternedMEAs were assessed for over 2 weeks. The cells attached to thefibronectin, but not to the PEG background and showed clearly delineatedregions (FIG. 1). This allowed the beating cells to grow exclusivelyover specific electrodes. The cells formed monolayers by day 2 andspontaneous contraction and beating activity began on day 4 and wasconsistent throughout the pattern. Cell survival and activity improvedmarkedly with the addition of L-thyroxine, Epidermal growth factor andHydrocortisone to the culture medium as indicated in the Methods. In aprevious publication cells were shown to maintain spontaneous beatingactively for up to 2 months in vitro [15]. Additionally, and by way ofprophetic example, we believe the cardiomyocytes used in the inventioncould be derived from human embryonic stem cells (HESCs).

Extracellular Recordings from Patterned Cardiomyocytes on the MEAs

The electrical activity of the spontaneously contracting cells patternedon the microelectrodes was extracellularly recorded using the MEAsystem. Cardiac myocytes formed a morphologically homogenous, aligned,integrated network of cells that communicated through gap junctions anddisplayed normal tissue electrophysiology. The recorded field potential(FP) signals correlated with the contraction cycle.

In random cultures the direction and speed of the propagation ofexcitation (action potentials) have been shown to depend on a number ofvariables, including intercellular resistance across the gap junctionsand the depolarizing sodium current[43-45]. For extracellular MEArecordings, previous research had indicated that the excitation wavecould be followed through the monolayer as the increasing delay in fieldpotential peak times and could be used to determine the pathway of theexcitation as it moved away from the initiation site[46]. However, adirect determination of the CV has been difficult in unpatternedmonolayers, even though the timing of FP generation could be estimated.With patterning of the cells on the electrode array, the exact path of aspontaneous excitation wave can be determined and then, using the pathlength, conduction velocity can be calculated with a high degree ofaccuracy. Moreover, because of the pre-determined pathway, there is noneed to image the wave propagation, just the measurement of the startand end times of the waves on the patterns as recorded by themicroelectrodes in the array. Thus, in later high-throughputapplications significantly fewer electrodes would be necessary for thesemeasurements and imaging would not be a necessary requirement foranalysis. FIG. 2 shows a typical recording and the method for thedetermination of propagation direction. FIG. 2B emphasizes the delaybetween FPs at different electrodes on the pattern, which enabled thedetermination of CV with high temporal resolution. The signalpropagation was also visualized using an Imaging software routinewritten in Matlab 6.5[47]. The program converted recordings from theelectrodes into a video indicating the field potential movement acrossan 8×8 grid. Snapshots from the video are shown in the supplementarydata. This program also verified the origin of the excitation wave andconfirmed our CV results obtained using the earlier method (FIG. 2.).

The conduction velocity was calculated to be 0.190±0.025 m/s forspontaneous firing of the patterned cardiomyocytes over eight differentMEAs. Previous research has shown that in vitro conduction velocitiesranged from 0.12 m/s[48] to 0.242 m/s[49]. In a computer simulation ofAP propagation in cardiac fibers in realistic conditions (taking intoaccount extracellular solutions and spaces) the conduction velocity was0.504 m/s[50]. In the human heart the AP generated by the Sino-atrialnode spreads through the atria at a conduction velocity of 0.5m/sec[51].

The conduction velocity on patterned cardiac monoloyers was alsomeasured with stimulation. In these experiments, the cardiac pattern wasstimulated at the two ends of the pattern, over a distance of 0.77 mmwith 1 ms wide bipolar pulses with an amplitude of 500 mV at a frequencyof 2 to 4 Hz (FIG. 3). The conduction velocity at 2 Hz stimulation wascalculated to be 0.315±0.011 m/s (n=8). Previous research has indicatedthat rapid electrical stimulation in cultured, neonatal ratcardiomyocytes had an effect on the expression of Connexin [43] leadingto changes in conduction properties[52], including an increase inconduction velocity.

This study showed that rapid electrical stimulation caused an increasein conduction velocity compared to the non-stimulated or spontaneouscase. These results also indicated that accurate estimation ofconduction velocity and propagation path could be determined in both thestimulated and in the spontaneous case. The measurement of the frequencydependence of conduction velocity could help in the understanding of theelectrophysiological properties of cultured, neonatal rat cardiacmyocytes, and ultimately, the physiology and pharmacology of the heart.The stimulated conduction velocity was also closer to the modeled valuesin the literature as well as to those generated by the SA node in theheart in vivo.

Electrical stimulation optimization experiments and measurement ofrefractory period after the AP The stimulation protocol was optimized bydetermining a minimal threshold which reliably generated contractionfrom the cells. The cardiomyocytes were stimulated with 2 Hz, 500 mV, 1ms wide bidirectional pulses at different electrodes. As seen in FIG. 3,the time-delay between the stimulation artifact and the FP at thedifferent electrodes is in good correlation with the distance betweenthe stimulation and recording electrodes. The excitation wave spreadevenly on branched patterns as evidenced by the zero time difference atthe end of the branches.

As shown in FIG. 4, paired pulse stimulation protocols could also beused with the defined cardiac patterns. By varying the time-delaybetween stimulations the absolute/relative refractory period after APgeneration could be mapped (FIG. 4). If the stimulation delay betweenthe two electrodes was too short, the second stimulus failed to evoke asecond propagating AP. This experiment suggests new possibilities tostudy phenomena difficult to measure in random (not constrained)monolayers, such as re-entry, the refractory period after an AP andarrhythmia.

Pharmacology and Toxin Studies

One of the most important applications of this system could be themeasurement of the effect of drugs and toxins on cardiac function. Twopharmacological agents, 1-Heptanol and Sparfloxacin, were tested todemonstrate the usability of this system for pharmacological andtoxicology studies.

Patterned spontaneously beating cardiomyocytes were exposed to 50 μM1-Heptanol in the recording medium and the FPs were recorded at 15minute intervals. A control recording was taken before the toxinapplication. As shown in FIG. 5, 1-Heptanol had a marked effect on thetime for propagation of the excitation wave; it significantly increasedthe value measured before treatment. The increase in the time delayindicates a block and uncoupling of the gap junctions and delays the FPbetween the two recording electrodes. The CV was calculated to be0.0066±0.0004 (n=4), a significant decrease from 0.197±0.014 m/s (n=4),and this result clearly shows that the effect of a gap junction blockercan be analyzed using this system.

Effect of Sparfloxacin

Sparfloxacin is a fluoroquinone antibiotic and HERG channel antagonistthat is known to cause QT prolongation, Tdp and ventricularfibrillation[13, 53]. Application of 2 μM of Sparfloxacin to therecording medium caused a significant change in the spontaneous beatingfrequency within 25 minutes, with an increased FP length,non-synchronous contractions similar to fibrillation and burst-likebeating activity as shown in FIG. 5. The beating frequency changed froma stable value of 0.6 Hz-1 Hz to intra-bursts with a higher frequencyrange of 1 Hz-1.2 Hz. The loss of synchrony between the electrodesmeasured also indicated a propagation block in the monolayer.

Discussion

Surface Modification, Photolithographic Patterning and Patterned CardiacMyocyte Cultures on Microelectrode Arrays

In this study, we have shown that the surface of commercialmultielectrode arrays could be functionalized with SiPEG self-assembledmonolayers. The PEG layer may be ablated and subsequently patternedusing photolithographical techniques and that incubation of the patternswith 5 μg/ml fibronectin for 20 minutes did not affect the cellresistive properties of PEG, but significantly improved the attachmentof cardiac cells to the glass surface. Surface analysis of the MEAsafter modification indicated that both PEG-Silane modification andfibronectin adsorption on the ablated areas of the MEA surface wassuccessful. The patterned commercial electrodes were reusable 6-10 timeswithout observable decline in the quality of electrophysiologicalrecordings. Rat neonatal cardiac myocytes demonstrated normal physiologyand formed beating monolayers on the areas designated by the patterns.

Manipulating cells, determining their attachment, growth anddifferentiation in cultures has became important in applications such astissue engineering, toxin detection, drug screening and robotics.Unfortunately, there is no generally applicable method to pattern allcell types. The method developed here using a protein adsorptionresistant background, SiPEG, followed by protein adsorption(fibronectin) to the ablated area is a relatively simple, fast andinexpensive solution for this problem and could be generalized for‘difficult to pattern’ cells.

Electrophysiology, Toxin and Drug Effects

The patterned cells formed a confluent, aligned and interconnectednetwork of spontaneously contracting cells communicating via gapjunctions. The activity of the cells could be measured using the MEA.The extracellular electrodes allowed not only recording, but alsostimulation of the patterned cardiac myocyte monolayers. For themeasurement of conduction velocity three different methods were used: 1)traditional video-monitoring of the excitation wave in the monolayer, 2)measurement of the speed of the spontaneous excitation waves on thepatterns via the MEA and 3) measurement of the propagation of astimulation-evoked extracellular field potential. All of these methodsproduced consistent results which were in reasonable agreement withpublished in vivo data. Conduction velocity increased with rapidelectrical stimulation indicating that cardiac cell physiology can bestudied using this method. Also, it was seen that the variability of theCV measurements decreased from about 15% as seen with the recordingsfrom spontaneous APs to about 5% with stimulated APs. Patterning of themonolayers made CV measurements simpler, faster and more reliable. Infuture commercial applications this may be a more cost effective and ahigher throughput technique for screening the cardiac side effects ofdrug candidates as it also eliminates the need for imaging of thepropagation waves in the system. Patterning would also drasticallyreduce the number of required electrodes.

We have also demonstrated that alternating stimulation at differentelectrodes on the patterns could be used to measure the refractoryperiod after APs, an important parameter for determining potentiallyfatal pharmacological side effects. This technique could also be avaluable tool for the study of cardiac defects, such as reentrantarrhythmia. Further development of this technology, especially for usewith human cells, could significantly affect pharmaceutical drugdevelopment enabling high information content cardiac side effectscreening.

Two pharmacological agents, 1-Heptanol and Sparfloxacin, were used todemonstrate the effectiveness of this method for drug screening. Both ofthese compounds are known to have cardiac side effects. 1-Heptanol is agap junction blocker, whereas Sparfloxacin is an antibiotic that wastaken off the market due to cardiac side effects. Our results were inagreement with the literature as 1-Heptanol drastically decreased the CVin the cardiac myocyte monolayers without completely blocking theconduction, whereas Sparfloxacin caused fibrillations which initiated acomplete conduction block. These results indicated that specific drugactions can be studied using this system. 1-Heptanol affected theconduction velocity properties, while maintaining the synchronicity ofthe beating cells. The addition of Sparfloxacin to the cells caused aloss in rhythmicity resulting in a burst-like activity similar tofibrillations that are shown to have occurred in animal and human trialsfor the drug. These promising initial results support the need forfurther development of this system as a high-throughput,high-information content functional drug screening platform.

CONCLUSION

A simple and reliable method for patterning cardiac myocytes onmultielectrode arrays has been disclosed. This method is compatible with1.5 standard silicon manufacturing steps. Patterning of the cardiacmyocytes increased the information content of the traditionalmultielectrode array recordings by enabling measurement of CV in a fastand simple way. Moreover, using paired stimulation, the measurement ofthe refractory period after the action potentials became possible.Measurement of the effect of drugs with known cardiac effects, was inagreement with the literature. Further development of this method couldresult in cheaper, faster, pharmaceutical side-effect screening withhigher predictive value.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

REFERENCES

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That which is claimed:
 1. An in vitro method of testing the effect of acompound on cardiac function, the method comprising: a) providing aculture of patterned spontaneously beating cardiomyocytes comprising: i)a support substrate bearing a multielectrode array; ii) a first surfaceresistant to cell attachment and deposited on said support substratewherein the first surface covers the multi-electrode array; iii) apattern ablated on said first surface; iv) a second surface thatpromotes cell attachment deposited on the pattern ablated on the firstsurface; and v) cardiomyocytes adherent to said second surface andgrowing aligned along said pattern to provide said culture of patternedspontaneously beating cardiomyocytes; b) recording a first electricaloutput from said culture of patterned spontaneously beatingcardiomyocytes; c) contacting said culture of patterned spontaneouslybeating cardiomyocytes with a compound being tested; d) recording asecond electrical output from said culture of patterned spontaneouslybeating cardiomyocytes after contacting with the compound; and e)determining a change between the first and second electrical outputs asan indicator of altered cardiac function.
 2. The method of claim 1,wherein the first surface comprises a material selected from the groupconsisting of polyethylene glycol, polyacrylic acid and polyacrylamide.3. The method of claim 1, wherein the material is polyethylene glycoldeposited essentially as a monolayer.
 4. The method of claim 1, whereinthe pattern is ablated by photolithography.
 5. The method of claim 1,wherein said culture of patterned spontaneously beating cardiomyocytesare of mammalian origin.
 6. The method of claim 1, wherein said cultureof patterned spontaneously beating cardiomyocytes are of rat origin. 7.The method of claim 1, wherein said culture of patterned spontaneouslybeating cardiomyocytes are derived from human embryonic stem cells. 8.The method of claim 1, wherein the second surface comprises fibronectin,trimethoxysilylpropyldiethylenetriamine, or pure glass.